Requirement for N-ethylmaleimide-sensitive factor activity at different stages of bacterial invasion and phagocytosis

Bacterial invasion, like the process of phagocytosis, involves extensive an d localized protrusion of the host cell plasma membrane. To examine the mol ecular mechanisms of the membrane remodeling that accompanies bacterial inv asion, soluble NSF attachment protein receptor (SNARE)-mediated membrane tr affic was studied in cultured cells during infection by Salmonella typhimur ium. A green fluorescent protein-tagged chimera of VAMP3, a SNARE character istic of recycling endosomes, was found to accumulate at sites of Salmonell a invasion. To analyze the possible role of SNARE-mediated membrane traffic in bacterial infection, invasion was measured in cells expressing a domina nt-negative form of N-ethylmaleimide-sensitive factor (NSF), an essential r egulator of membrane fusion. Inhibition of NSF activity did not affect cell ular invasion by S. typhimurium nor the associated membrane remodeling. By contrast, Fc gamma receptor-mediated phagocytosis was greatly reduced in th e presence of the mutant NSF. Most important, dominant-negative NSF signifi cantly impaired the fusion of Salmonella-containing vacuoles with endomembr anes. These observations indicate that the membrane protrusions elicited by Salmonella invasion, unlike those involved in phagocytosis, occur via an N SF-independent mechanism, whereas maturation of Salmonella-containing vacuo les is NSF-dependent.